Knowledge of the mechanisms that modulate glucose-stimulated insulin secretion (GSIS) by pancreatic beta cells, particularly in the face of insulin resistance, is of paramount importance for understanding the pathogenesis of type 2 diabetes mellitus. Over the previous project period, it has become clear that even small changes in glucokinase (GK) activity in the pancreatic beta cell affect GSIS, that insulin action in the beta cell modulates GSIS, and that insulin affects GK gene expression, both at the transcriptional and post-translational levels. This proposal will explore several aspects of GK regulation and function. We will continue to place an emphasis both on the pancreatic beta cell, since it plays a principal role in most glycemic disorders and serves as a model for other glucose-sensitive cells, and on the generation of novel new mouse models. However, some studies will involve other sites of GK expression, such as the brain, in order to learn more about how the expression of GK in certain neurons contributes to the regulatory feedback loops that control the blood glucose concentration. In Aim 1 we will determine whether GK is necessary for neural glucose sensing by generating and characterizing brain-specific GK gene knockout mice. In Aim 2 we will establish true mouse models for PHHI and MODY-2 in order to correlate changes in enzyme kinetics with insuIin secretion and glucose homeostasis. In Aim 3 we will determine the role of alternate RNA processing the insulin receptor in modulating both GK gene transcription and subcellular localization in the pancreatic beta cell.